A variety of cytotoxic proteins have been derived from plant and bacterial sources. One such protein, trichosanthin, is an abortifacient protein extracted from the roots of Trichosanthes kirilowii (Shanghai Inst. Organic Chem., Nucleic Acids and Proteins, Proc. Symp. Nuc. Acids and Proteins, Van Nostrand Reinhold Co., 1979, 318-323). T. kirilowii is a plant which is indigenous to the Orient, and has been used in crude form in traditional Chinese medicine for centuries The protein is also known as Radix trichosanthes. It is a basic protein consisting of a single polypeptide chain of Mr 25,000 Da containing approximately 219 amino acid residues (Kezhan et al., Scientia Sinica 25: 730-737, 1982). The abortifacient activity of trichosanthin is apparently mediated through an effect on placental trophoblast cells (Shanghai Inst. Exp. Biol. Second Lab, Scientia Sinica 19: 811-827, 1976). Additional therapeutc applications of trichosanthn include treatment of ectopic pregnancy, benign and malignant hydatidiform mole and choriocarcinoma. Use of trichosanthin in treatment of choriocarcinoma is consistent with its abortifacient activity, as this uterine cancer is derived from fetal trophoblast cells. The abortifacient activity of trichosanthin is discussed by Cheng Kuo-Fen (Obstet. Gynecol. 59: 494-498, 1982). However, the mechanism of action of this protein has not been elucidated.
Yeung et al. (Int. J. Peptide Protein Res. 27: 325-333, 1986) purified three lectins from the roots of Trichosanthes kirilowii. These were shown to have erythroagglutinating activity, but not abortifacient activity.
As noted above, various cytotoxic proteins derived from plant and bacterial sources have also been described. Plant-derived toxins include abrin, isolated from Abrus precatorious, and ricin, isolated from Ricinus communis. Bacterial toxins include diptheria toxin and pseudomonas exotoxin. These molecules are structurally and functionally similar, being composed of a B-chain, which recognizes and binds to cell surface receptors, the B-chain being disulfide bonded to an A-chain which enters the cell and interferes with protein synthesis. A single molecule of such a toxin may be sufficient to kill a cell.
An additional class of cytotoxic proteins isolated from plants is exemplified by gelonin, an inhibitor of protein synthesis extracted from the seeds of Gelonium multiflorum (Stirpe et al., J. Biol. Chem. 255: 6947-6953, 1980). Unlike ricin and similar toxins, gelonin consists of a single polypeptide chain and does not exhibit generalized cytotoxic effects, apparently due to an inability to bind to and enter intact cells.
Recently, there has been considerable interest in producing therapeutic agents consisting of a cytotoxic protein coupled to a targeting agent such as an antibody (see review by Thorpe and Ross, Immunological Rev. 62: 119-158, 1982) or other protein such as a growth factor, which reacts with specific receptors on cells (Pastan et al., U.S. Pat. No. 4,545,985). Through the use of tumor-specific antibodies or other proteins that recognize markers which are highly expressed on the surface of tumor cells, these conjugates should exhibit specificity to tumor cells. However, these efforts have led to mixed results. Conjugates which comprise intact two-chain toxins may lack specificity due to their residual ability to bind nonspecifically to cells via the B-chain. Attempts to modify the toxin chemically to prevent nonspecific binding are not always successful and may reduce the activity of the A-chain. Conjugates using A-chain alone often have significantly lower potency as compared to whole toxin conjugates, apparently due to the role of the B-chain in mediating the entry of the toxin into the cell (Thorpe and Ross, ibid; Vitetta and Uhr, Cell 41: 653-654, 1985 and references therein). Furthermore, the use of ricin A-chain alone in an immunotoxin reduces generalized cytotoxicity by only one order of magnitude.
It is clear that many currently available immunoconjugates are unsuitable for cancer therapy due to a lack of specificity or lack of potency. Therefore, there is a need in the art for cytotoxic agents which by themselves lack generalized cytotoxicity, but which may be coupled to a targeting agent to produce a highly specific toxin conjugate. The present invention provides such a cytotoxic agent while further providing additional related advantages.